Chasing allosteric inhibition of the SARS-CoV-2 PLpro via molecular dynamics simulations with flooding fragments (MDFFr)

The SARS-CoV-2 papain-like protease (PLpro) represents a crucial therapeutic target due to its dual role in viral polyprotein processing and suppression of host immune responses through de-ubiquitination and de-ISGylation activities. To identify novel allosteric druggable sites on PLpro, we developed a molecular dynamics approach with flooding fragments (MDFFr), which extends a previously established method –Molecular Dynamics flooding– enabling broader applicability across biological targets. Using MDFFr, we evaluated interactions of known phenolic inhibitors with SARS-CoV-2 PLpro and identified several biologically significant sites, encompassing allosteric hotspots, cryptic pockets, and regions involved in protein–protein interactions. Our simulations not only confirmed experimentally characterized binding sites, including fragment-binding and protein–protein interaction regions for ubiquitin and ISG15 (Interferon-Stimulated Gene 15), but also uncovered previously unrecognized hotspots for further investigation. These results establish MDFFr as a suitable approach for physics-based druggability assessment of biological targets using only protein 3D structure, while providing detailed insights into fragment-protein interactions at both druggable sites and protein–protein interfaces. These findings also unveil new opportunities for allosteric inhibition of PLpro, potentially advancing therapeutic strategies against SARS-CoV-2 and other coronavirus-related diseases. Furthermore, by using “real” drug-like fragments (rather than standard cosolvent “probes”), MDFFr enhances translational relevance and directly informs drug repurposing and ligand discovery efforts.